The broad objective of this proposal is to examine the roles of cdx genes and retinoic acid (RA) in regionalizing the zebrafish midgut, with particular emphasis on the pancreas and liver. RA signaling has been shown to be necessary to specify the zebrafish pancreas and liver, and the role of RA in pancreas development is conserved across vertebrates. The mechanism whereby RA functions in this context is unknown. Studies on mesoderm and ectoderm have suggested that RA functions upstream of Cdx transcription factors, which in turn signal to downstream hox genes. My preliminary experiments have established that cdx4 is required for normal pancreas localization along the digestive tract. My general strategy is to test the working hypothesis that pancreas localization relies on integrating anteriorally-derived RA signals and posteriorally-derived Cdx signals. I will investigate the roles of cdx4 and cdx2 in regionalizing the endoderm using loss-of-function approaches. Since Cdx genes have been shown to function synergistically in other systems, I will investigate whether cdx4 and cdx2 function synergistically during endoderm patterning. Using cell transplantation I will generate chimeric embryos to determine whether cdx4 influences pancreatic fate cell-autonomously versus non-cell-autonomously. Finally, I will investigate the role of RA signaling in regulating Cdx genes utilizing loss-of-function approaches as well as treatment with exogenous RA.